Until quite recently, solid-phase methods for the synthesis of peptides or proteins have to a large extent been based on the original methodology developed by Merrifield, employing a functionalized cross-linked styrene/divinylbenzene copolymer, the cross-linked copolymer having been formed by the polymerization of styrene monomer to which has been added a few per cent (typically about 2%) of divinylbenzene. This copolymer is generally provided in the form of beads or particles, often with a dominant particle size of 20-80 .mu.m. The functionalization originally preferred by Merrifield see e.g. J. Am. Chem. Soc. 85, 2149 (1963)! was a functionalization of the aromatic rings of the copolymer with chloromethyl groups, introduced via reaction of the solid copolymer with SnCl.sub.4 /chloromethyl methyl ether, although a number of other functionalities, including aminomethyl, .alpha.-aminobenzyl and .alpha.-amino-4-methylbenzyl, have subsequently been employed. Regardless of its nature, the purpose of the functionality is normally to form an anchoring linkage between the copolymer solid support and the C-terminal of the first amino acid which it is desired to couple to the solid support. Later refinements of the Merrifield methodology have included the further introduction, between a functionality (e.g. one of the above-mentioned functionalities) on the polystyrene chains and the C-terminal of the first amino acid which is to be coupled, of a bifunctional "spacer" or "handle" group whose reactivity is tailored inter alia to meet desired requirements with respect both to the coupling of the first amino acid to the solid support and/or to the ease with which the completed, synthesized peptide or protein chain is cleaved from the solid support. Examples of such spacer groups include the phenylacetamidomethyl (Pam) and the p-alkoxybenzyl ester systems. Barany et al. Int. J. Peptide Protein Res. 30, 705-739 (1987)! have reviewed the development of solid-phase peptide synthesis methodology from its introduction by Merrifield up to about 1986.
The advances in biotechnology which have been made in the last decade or so, particularly in the area of recombinant DNA, have produced a situation in which vast numbers of new protein sequences with undefined or unknown function and/or unknown biological activity have become available. In this connection, detailed structural analysis by site-directed mutagenesis or similar molecular engineering techniques has provided a useful approach to the study of the roles of amino acid residues in active sites of proteins.
However, specific information concerning biologically active functional subunits (peptides) containing ca. 5-40 amino acid residues is preferably obtained through chemical synthesis. Whilst the chemical methodology which had been developed within the period reviewed by Barany (vide supra) was quite capable of yielding such peptides reliably and in high purity when using the above-outlined "conventional" method of solid-phase peptide synthesis (via a "linear" mode of approach), only one peptide was produced per synthesis.
A greater need than ever thus arose for methods employing a "simultaneous" or "parallel" mode of approach to the synthesis of peptides so that a large number of peptides could be synthesized simultaneously (or substantially simultaneously). These peptides could then be used, for example, to define and map the functional entities of proteins. The obvious advantages of a method permitting the parallel and substantially simultaneous synthesis of a multitude of peptides are the attendant saving in time, and the redundancy of the repetitive labour involved in accomplishing the synthesis of each peptide individually.
In this connection, a basic feature of the solid-phase technique of peptide synthesis is that in each elongation of the peptide chain with a further amino acid, all treatment steps are repetitive of the previous cycle with the possible exception of the amino acid coupling step itself, in which a further amino acid that may or may not be identical with that coupled in the preceding cycle is coupled to the peptide chain. Thus, parallel, substantially simultaneous synthesis of two or more peptides could be achieved by performing in parallel the repetitive steps, such as deprotection, neutralization, and washing, which are common to the parallel syntheses. The major technical difficulty here had been the attainment of compartmentalization of each amino acid coupling step so that cross-contamination did not occur.
Several different methods have been proposed for the substantially simultaneous synthesis of a number of peptides:
One of these methods Geysen et al., Proc. Natl. Acad. Sci. USA. 81, 3998-4002 (1984) and 82, 178-82 (1985)! was devised for rapid screening of peptide epitopes via ELISA (Enzyme Linked Immunosorbent Assay) in 96-microtiter wells. It utilizes acrylic acid-grafted polyethylene rod-and-96-microtiter wells to immobilize growing peptide chains and to perform the compartmentalized synthesis. However, while highly effective, the method is not applicable on a preparative scale, i.e. to the preparation of milligram quantities.
A second method Houghten, Proc. Natl. Acad. Sci. USA. 82, 5131-35 (1985)! utilizes a "tea bag" containing the traditionally used polymer beads to compartmentalize the synthesis, portions of peptidyl-resin beads being kept apart in sealed bags of fine-mesh polypropylene net. The latter method is relevant to the preparation of milligram quantities.
A more recent, and highly effective, third method is disclosed in WO 90/02749. The basic method of peptide synthesis disclosed therein uses a polymer substrate--especially a polyethylene substrate--which is preferably in the form of a sheet or film, and to which polystyrene chains have been grafted. The polystyrene chains, which may bear further substituents which are not reactive under the conditions prevailing in the synthesis, have an estimated peak molecular weight (not including optional substituents) of at least 200,000, and at least part of the grafted polystyrene chains are functionalized with a chemical functionality which facilitates the formation of an anchoring linkage between the polystyrene moiety and an at least N-protected and optionally carboxyl terminal derivatized amino acid.
Very high loading with synthesized peptide can be achieved with a polystyrene-grafted polymer substrate as disclosed in WO 90/02749, and when in the preferred form of a sheet or film, such a polystyrene-grafted polymer substrate is particularly convenient for the purposes of parallel synthesis of multiple peptides. Thus, for example, it is easy to cut a sheet or film into pieces of any desired size and/or shape and to transfer such pieces, if appropriate, from one reaction vessel to another; furthermore, a material in the form of a sheet or film is easy to mark for identification purposes (e.g. in connection with the latter mentioned transfer between various reaction vessels).
Thus, when the invention disclosed in WO 90/02749 is employed in the context of parallel and substantially simultaneous synthesis of a plurality of peptides, an appropriate plurality of essentially identical substrates of the above-described type are provided, after which the synthesis proceeds as follows:
a) the members of the plurality of polystyrene-grafted polymer substrates are optionally physically segregated into two or more sets each comprising one or more members of the plurality; PA1 b) an N-protected and optionally carboxyl terminal derivatized amino acid is coupled to the functionalized polystyrene moieties of each member of the plurality or, where applicable, each member of each set, the N-protected and optionally carboxyl terminal derivatized amino acid employed being identical for all the members of the plurality or, where applicable, all the members of one set, and, where applicable, further being in accordance with one of the following alternatives: PA1 the functionality and the N-protected and optionally carboxyl terminal derivatized amino acid being adapted to each other such that the anchoring linkage formed can subsequently be cleaved substantially without degradation of the peptide or protein chain which is to be synthesized; PA1 c) each member of the plurality or, where applicable, each member of each set is treated so as to remove the N-protecting group from an N-protected amino or substituted amino group of the coupled and N-protected amino acid, such that reaction of the amino or substituted amino group of the coupled amino acid with a carboxyl group or an activated carboxyl group of a further N-protected amino acid is facilitated; PA1 d) the amino or substituted amino group of the amino acid last coupled to the functionalized polystyrene moieties of each member of the plurality or, where applicable, of each member of each set is reacted with a carboxyl group or an activated carboxyl group of a further N-protected amino acid, so as to form a peptide bond between the amino or substituted amino group and the carboxyl group or activated carboxyl group, the further N-protected amino acid being identical for all the members of the plurality or, where applicable, all the members of one set, and, where applicable, further being in accordance with one of the three alternatives mentioned above in connection with step b); PA1 e) each member of the plurality or, where applicable, each member of each set is optionally treated so as to remove the N-protecting group from an N-protected amino or substituted amino group of the last-coupled N-protected amino acid, such that reaction of the amino or substituted amino group of the latter amino acid with a carboxyl group or activated carboxyl group of a further N-protected amino acid is facilitated; PA1 f) in those cases where step e) has been performed, steps d) and e) are repeated a desired number of times; PA1 g) each member of the plurality or, where applicable, each member of each set is optionally treated so as to remove some or all protecting groups possibly remaining on the amino acid moieties of the synthesized peptide or protein chain; PA1 h) each member of the plurality or, where applicable, each member of each set is optionally treated so as to cleave the linkage anchoring the synthesized peptide or protein chain to the functionalized polystyrene moieties of each member of the plurality or, where applicable, of each member of each set; and, PA1 i) if appropriate, any further undesired group is removed from a synthesized peptide or protein chain. PA1 (i) still possesses excellent mechanical and optical properties, PA1 (ii) can withstand harsher chemical/physical conditions (e.g. higher temperatures, aggressive solvents) than the material of WO 90/02749, PA1 (iii) retains all the advantages of the material of WO 90/02749 with respect to, e.g., peptide synthesis, and PA1 (iv) can be further modified by functionalisation or derivatization with appropriate substituent groups so as to acquire a high degree of hydrophilicity while at the same time being compatible with synthetically relevant organic solvents and retaining good optical and mechanical properties.
(i) identical for all the sets, PA2 (ii) when the number of said sets is greater than two, identical for at least two of the sets, PA2 (iii) different for each set,
The methodology disclosed in WO 90/02749 is equally applicable to the synthesis of a single peptide or protein. In this case only one polystyrene-grafted polymer substrate of the type in question is used, and the alternatives mentioned under b) and f), above, do not then apply; apart from this, the steps in the process are entirely analogous to those recited above.
It is also appropriate to mention here another document, viz WO 91/13098. This document discloses solid supports which are very closely related to the polystyrene-grafted substrates of WO 90/02749 and which are suited for use in solid-phase biosystems, such as bioassays, e.g. immunoassays. The solid supports of WO 91/13098 differ from those of WO 90/02749 only in that they are functionalized with chemical functionalities from which a peptide which has been attached thereto, or a peptide which has been synthesized thereon (e.g. in the manner disclosed in WO 90/02749), will not become detached under any normally applying chemical or physical conditions.
After a number of years of experience in the handling and use of the inventions described in WO 90/02749 and WO 91/13098, it has become apparent that although the substrate/support materials disclosed therein offer a number of significant advantages in relation to the state of the known art at the time of filing of the respective PCT applications, it would nevertheless be advantageous to be able to improve the properties of the materials in certain respects.
Thus, for example, it became clear that the occlusion, in the PE film material, of polystyrene homopolymer during the grafting process led to undesirable inhomogeneity of the grafted material owing to the formation of small bubbles or blisters in the material. Apart from the fact that it is necessary to carry out a time-consuming extraction of this occluded homopolymer from the PS-grafted PE film in order to obtain a material with satisfactorily reproducible chemical and physical behaviour in the context of applications to synthesis, the inhomogeneity due to the above-mentioned bubbles/blisters results in the optical properties (e.g. clarity, light transmission and reflecting properties) of the PS-grafted PE-material being considerably poorer than could be desired.
It has also been found that, for example, at high levels of loading of synthesized peptide or protein on the PS-grafted PE substrates of WO 90/02749, the mechanical properties of the material deteriorate, leading to a tendency for the material to fragment in an uncontrolled fashion.
Furthermore, the thermal and other properties of the PS-grafted PE substrates of WO 90/02749 impose limits on the nature of the functionalities which can be introduced into the polystyrene part of the material, and on the conditions under which the materials in question can be used (thus, for example, the PS-grafted PE material of WO 90/02749 dissolves in solvents such as xylenes at 100.degree. C.). The ability to be able to withstand functionalisation conditions entailing, for example, the use of highly "aggressive" organic solvents (such as xylenes) and relatively high temperatures would permit functionalisation with a range of functionalities which could considerably broaden the synthetic applicability of the substrate material, and would therefore constitute a highly desirable improvement of the material.
Moreover, in continuing investigations of the applicability of the substrates disclosed in WO 91/13098 (vide supra) for use in solid-phase biosystems, such as bioassays, e.g. immunoassays, it became more and more clear that the lack of hydrophilicity of the material imposed limits on its breadth of applicability in connection with, for example, assays of the ELISA type. It thus became apparent that a material of the general type in question but having significantly improved hydrophilic properties would have considerable advantages.